Apparatus and Method for Optical Sampling in Miniature Bioprocessing Vessels

ABSTRACT

An optical sampling apparatus for miniature-scale bioprocessing vessels includes features for optical interrogation of the bioprocessing vessel contents by means of transmission or transflection spectroscopy. This optical interrogation allows for the determination of quantities and parameters of substances in fluids contained within the bioprocessing vessels during bioprocesses. Multiple such bioprocessing vessels with the optical interrogation features may be mounted in a receiver for conducting multiple bioprocesses simultaneously. A translatable probe may be used to interact with each of the bioprocessing vessels in the receiver.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication No. 61/992,735, filed May 13, 2014, for “Optical Interfacesfor Bioprocessing Vessels.” Such application is incorporated herein byreference in its entirety.

BACKGROUND

The present invention relates to optical sampling means for providingoptical communication between an optical instrument and a disposablevessel of polymeric construction for applications including, but notlimited to, pharmaceutical, food processing, and chemical manufacturingas well as other laboratory and industrial processes.

The use of optical and electronic instrumentation to monitor and controlthe contents of vessels and changes taking place therein is well knownin the art. Processing and storage of, for example, food, beverage,chemical, agricultural, fuel, and pharmaceutical products havehistorically taken place primarily in multiple-use vessels comprised ofstainless steel and/or glass. Numerous hardware approaches enablinginterrogation and analysis of the contents of such vessels by, forexample, optical, electronic, and electrochemical techniques have beendescribed in the art. Dissolved oxygen may be measured by, for example,electrochemical probes with oxygen-permeable membranes, as well asfluorescent sensor techniques. Measurement of pH is possible byelectrochemical techniques as well as fluorescent methods. Probes formeasurement of optical characteristics of materials in rigid vessels bytransmission, reflection, and attenuated total reflection (ATR) are alsoknown in the art. Such probes are often of tubular form and primarilymetal construction, protruding through a head plate or side wall of avessel and into the fluid under process. Probes and sensors of thisgeneral description are commonly designed for robustness andlongevity—tolerating use, cleaning, and often sterilization for manyprocess cycles. Such multiple-use probes and sensors typically have formfactors that are not accommodating to interfacing with single-usebioreactors, particularly flexible bioreactors and those with smallworking volumes. Flexible bioreactors, also known as bag bioreactors,lack rigidity—surfaces commonly distort during operation, makingattachment and positioning of typical multiple-use probes difficult andunstable. Bioreactors with small working volumes simply do not have thesurface area or volume to support many of the sensors and probes thatare common in the art. Moreover, such prior art sensors and probes donot commonly fit within the model of single-use technology as they arenot disposable and must be in contact with the process fluid, therebyrequiring the cleaning, sterilization, and aseptic insertion steps thatsingle-use technology seeks to avoid.

Regular cleaning and maintenance of multiple-use vessels is required tomaintain process integrity, and sterile conditions are often necessary,demanding yet more laborious and/or costly cleaning and sterilizationprocedures. The maintenance, cleaning, and disinfection of multiple-useprocess vessels coupled with the high initial cost of the equipment hasled to accelerating adoption of single-use, disposable vessels inmultiple industries. These single-use vessels are most commonlyconstructed of polymers and are often purchased pre-sterilized such thatthe user may immediately put them to use. As such, sensors that willcome into contact with the fluid are commonly integrated into the vesselbefore sterilization and sterilized with the vessel. Any sensors orconnections to the vessel that are not integrated and sterilized withthe vessel may be externally sterilized and installed via aseptic ports.While use of sensors or probes that are not installed into the vesselprior to sterilization of the vessel is feasible, it is typicallyundesirable due to the additional labor required of the end user as wellas the increased probability of contamination. Such single-use vesselsoffer several additional benefits over conventional multiple-usebioreactors: ease of use; reduced setup labor for end users;significantly reduced cleanup time; and lower equipment costs.Single-use disposable bioreactors are available in a variety of sizesand form factors—working volumes range from sub-milliliter to thousandsof liters.

A key aspect in bioprocessing is being able to transition processes fromsmall-scale experiments in the research lab to a large-scale productionenvironment. The research and effort to transition from small-scaleexperiments to production is known as scale-up, and this process iscommonly challenging and time consuming. Scale-up often comprises threemajor phases—the research phase where initial studies are performed andprocesses are selected and verified; the pilot plant phase whereprocesses are further studied, refined, and verified in higher volumeprocesses; and the production phase where large-scale manufacturing isperformed. The conditions present in small-volume research bioreactorsmay be markedly different from those present in the larger bioreactorsin the pilot plant and on the production floor. Indeed, processes canvary considerably even between different bioreactors in the researchlab. In order to execute the scale-up process in the most efficientmanner possible, it is desirable to have the ability to optimize aplurality of process parameters and constituent concentrations, andoften to be able to control such parameters and constituentconcentrations. Ideally such monitoring and control capabilities will beuniform throughout the various stages of scale-up. Bioreactors havingworking volumes of microliters to few milliliters are commonly known asmicro-bioreactors, and are often configured such that multiplemicro-bioreactors are used to perform experiments in parallel. Suchmultiplexed experiments with cell culture or fermentation processesenable evaluation of process conditions, cell lines, or other variablesin an efficient manner. So-called miniature-bioreactors commonly haveworking volumes of tens to few hundreds of milliliters, and may offeranother step in the scale-up process. Similarly to micro-bioreactors,mini-bioreactors are often configured in groups for parallelexperimentation, though with a working volume that better representsmore standard process conditions. While reliable monitoring ofconstituent concentrations of fluids in bioprocesses such as nutrientanalyte concentrations remains challenging even in large-volumebioreactors, the challenge is amplified with micro- and mini-bioreactorsgiven the space constraints and form factors. Sensor technologiescapable of providing such fluid constituent concentration information,and ideally control of such concentrations, in bioreactors used acrossthe product development arc from research lab to production plant aredesired in the biotechnology and pharmaceutical industries.

Sensors for measurement of a variety of parameters within single-usevessels have been demonstrated. For example, analysis of physical andchemical conditions such as pH and dissolved oxygen (DO) is possible bymeans of sensors comprising fluorescent dots within the bioreactorfluid. Single-use and disposable temperature and pressure sensors havebeen demonstrated. Optical interfaces for vessels of polymericconstruction, which may be single-use and/or flexible vessels, are alsoknown in the art, though to a far lesser extent than similar interfacesfor multiple-use vessels. Interfaces for transmission, reflection, andATR optical measurements have been disclosed; however these interfacesand ports are generally not optimized for near-infrared spectroscopicapplications. Numerous polymers are available that are at leastpartially transparent to visible and short-wave infrared (SWIR), thoughthese polymers are often substantially opaque or exhibit significantabsorption structure at wavelengths longer than 1.5 μm.

Bioreactors commonly require frequent monitoring and strict control inorder to ensure optimal environmental and nutritional conditions forfermentation, cell cultures, or similar processes contained therein.While sensors are available to continuously measure parameters such asDO and pH as is hardware and software to control these parameters,sensors and systems to monitor nutrients and chemical constituents in anautomated fashion and control the levels thereof have historically beenlargely absent in the art. This is the case for both multiple- andsingle-use bioreactors, however sensor solutions to interface withsingle-use bioreactors have been particularly lacking.

Measurement of chemical constituents by spectroscopic methods,particularly infrared spectroscopic methods, presents a robust means tomonitor said chemical constituents and control levels thereof withinbioreactors and process vessels in general. In order to opticallyinterface with polymeric vessels and their contents, integrated androbust optical interface solutions are desired. These solutions may besubstantially transparent in the wavelength range of interest, therebyenabling high measurement stability and optical throughput. Therequirement of material transparency is particularly challenging forinfrared spectroscopy, principally near- and mid-infrared spectroscopy,where optical absorption by many commonly used polymers is unacceptablyhigh when polymer thicknesses are within the satisfactory range tomaintain mechanical integrity. When in an optical spectroscopicconfiguration, embodiments of optical sensors where the path or samplelength through the vessel contents is selectable and/or controlled maybe desirable for some applications. Embodiments where any opticalelements that are to come in contact with the vessel contents are fusedto the vessel and sterilized with the vessel are often preferable tosolutions where optical monitoring components are inserted asepticallysubsequent to sterilization.

BRIEF SUMMARY

As used herein, the terms “optical” and “light” refer to electromagneticradiation having vacuum wavelengths between 300-20,000 nm.

As used herein, “near infrared”, “near-infrared”, and “NIR” mean theregion of the electromagnetic spectrum generally spanning wavenumbersbetween 3300 cm⁻¹ and 14,000 cm⁻¹ (corresponding to wavelengths ofapproximately 0.7 μm to 3.0 μm).

As used herein, “interrogation” and “sampling” mean illuminating asample with optical radiation and collecting at least a portion of theradiation having interacted with said sample for optical analysis.

As used herein, “working volume” refers to the typical volume of fluidcontained within a vessel or container during a process and is mostcommonly less than the total volume of fluid that the vessel couldretain.

As used herein, “miniature,” and “mini,” when used in reference tobioprocessing vessels means bioprocessing vessels having working volumesless than or equal to 0.25 liters.

As used herein, “constituent” means a chemical analyte, protein, DNA,component in a fluid, cell, or solid suspended in a fluid.

The present invention relates to miniature-bioprocessing vesselscomprising features for optical interrogation of fluids contained withinsuch bioprocessing vessels. Embodiments of receiver assemblies forreceiving, housing, and positioning embodiments of such bioprocessingvessels are also provided. Embodiments of such receivers may alsoprovide optical elements, sensors, and means for optical communicationwith optical instruments, and may be configured to receive a pluralityof bioprocessing vessels. An optical instrument may be used inconjunction with embodiments of the present invention to determineand/or control quantities of substances in fluids contained withinbioprocessing vessels. The invention pertains to optical transmissionand transflection measurements in general and particularly tonear-infrared spectroscopic analytical techniques.

A plurality of embodiments of bioprocessing vessels comprising featuresfor optical interrogation is described herein. Embodiments ofbioprocessing vessels provided by the present invention typicallycomprise at least one rigid polymer sidewall and may be comprisedentirely of rigid polymer materials. In such embodiments, at least aportion of the polymeric vessel may be substantially transparent to thewavelengths of electromagnetic radiation being utilized either due toinherent lack of absorption or to use of a suitably thin section ofpolymer. In one embodiment of the present invention, an optical samplingregion is provided whereby features extending outward from the primaryvolume of a bioprocessing vessel at least partially define the opticalsampling region by providing a defined length of optical path through afluid contained within the bioprocessing vessel. In another embodiment,an optical sampling region is provided whereby features extending intothe primary volume of a bioprocessing vessel at least partially definethe optical sampling region.

Embodiments are also described whereby bioprocessing vessels compriseintegral optical probes extending into the vessel. In some embodiments,optical waveguides and/or optical elements within an optical probecommunicate light into the fluid within a bioprocessing vessel where itmay interact with the fluid, and a portion of the light havinginteracted with the fluid may be communicated by additional opticalwaveguides and/or elements to a sensor or optical instrument. In oneembodiment, integral probes are provided where the input and outputoptical communication is provided on a single surface integrated withthe probe. In another embodiment, input and output optical communicationare provided on different sides of the optical probe coupled todifferent sides of a vessel. In yet another embodiment, only inputoptical waveguides and/or elements are provided, and light is sensed bya sensor in a receiver for the bioprocessing vessel.

The detailed description and drawings provided herein will offeradditional scope to certain implementations of the present invention. Itshould be understood that the described implementations are provided asexamples only. Those skilled in the art will recognize that numerousvariations and modifications of the described implementations are withinthe scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an isometric view of a disposable bioprocessing vesselhaving features for optical sampling that extend outward from theprimary volume of the vessel on the side of the vessel.

FIG. 2 shows a top sectional view of the bioprocessing vessel in FIG. 1and an associated optical reader.

FIG. 3 shows an isometric view of a disposable bioprocessing vesselhaving features for optical sampling that extend outward from theprimary volume of the vessel on the bottom of the vessel.

FIG. 4 shows a side sectional view of the bioprocessing vessel in FIG.3.

FIG. 5 depicts a polymer laminate comprising multiple polymer materials.

FIG. 6 shows a top sectional view of a disposable bioprocessing vesselhaving features for optical sampling that extend outward from theprimary volume of the vessel on the side of the vessel and include areflector.

FIG. 7 shows a side sectional view of a bioprocessing vessel havingfeatures for optical sampling that protrude into the primary volume ofthe vessel and form a single optical path length.

FIG. 8 shows a side sectional view of a bioprocessing vessel havingfeatures for optical sampling that protrude into the primary volume ofthe vessel and form two optical path lengths.

FIG. 9 shows a side view of a disposable bioprocessing vessel comprisinga second polymer in the optical sampling region.

FIG. 10 shows a top sectional view of the bioprocessing vessel in FIG.9.

FIG. 11 shows an isometric view of a disposable bioprocessing vesselwith an integral optical probe.

FIG. 12 shows a side sectional view of the bioprocessing vessel in FIG.11.

FIG. 13 shows a side sectional view of a disposable bioprocessing vesselwith an integral optical probe having an optical sampling regiontransverse to the axis of the probe.

FIG. 14 shows a side sectional view of a disposable bioprocessing vesselwith an integral optical probe providing an optical sampling regionbetween the end of the optical probe and the bottom of the bioprocessingvessel.

FIG. 15 shows an isometric view of a receiver for a plurality ofdisposable bioprocessing vessels with several disposable bioprocessingvessels installed therein.

FIG. 16 shows a detailed view of the isometric view of the receiver baseassembly in FIG. 15.

FIG. 17 shows a sectional view of the receiver base assembly of FIG. 15with three embodiments of disposable bioprocessing vessels installed.

DETAILED DESCRIPTION

An embodiment of a disposable bioprocessing vessel comprising featuresto provide an optical sampling region is shown in FIG. 1 and FIG. 2. Theisometric view in FIG. 1 shows a bioprocessing vessel 100 with featuresthat extend outward sideways from the primary volume of the vessel. Thefeatures extending outward from the vessel comprise a first surface 110and a second surface 120 that provide an optical sampling region 130.Additional surfaces may be provided as necessary to provide a particulargeometry of the optical sampling region 130. For example, a curved orstepped surface may be provided adjacent to the optical sampling region130 in order to promote adequate mixing of the fluid within the opticalsampling region 130 and within the entire vessel 100. At least one rigidwall 140 is provided, though the entire bioprocessing vessel 100 maycomprise rigid polymer materials. The geometry of the optical samplingregion 130 may be configured to best suit the chosen application. Formeasurements in aqueous solutions with near-infrared electromagneticradiation, the optical path defined by the distance between first 110and second 120 surfaces (distance through the fluid within the vessel)will preferably be between 0.5 mm and 2.0 mm. This range of optical pathlength provides acceptably low attenuation due to water absorption andsufficient interaction length with the fluid sample to providenear-infrared measurements of substances within the fluid. The opticalpath length is taken as the path length that the beam takes through thefluid and does not include the wall thicknesses of the first 110 orsecond 120 surfaces. A transmission or transflection measurement may beprovided by embodiments of the present invention. In a transmissionmeasurement configuration, electromagnetic radiation having interactedwith the fluid is communicated through the second surface 120 anddetected outside of the disposable bioprocessing vessel. In atransflection measurement, a portion of the electromagnetic radiationhaving been communicated through the first surface 110 is reflected backtowards the first surface 110 by the fluid and substances containedtherein, and a portion of the electromagnetic radiation havinginteracted with the fluid and having reached the second surface isreflected from the second surface 120 (or a reflective element 190thereon or therein, depicted in FIG. 6) towards the first surface 110.As electromagnetic radiation from the portions having been transmittedthrough and reflected from the fluid sample in the optical samplingregion 130 comprises the resulting radiation incident on the firstsurface 110 in the direction of arrowed line B, a transflectionmeasurement may be provided.

The polymer materials that comprise the first 110 and second 120surfaces in the optical sampling region 130 will preferably be materialsbeing sufficiently transparent to the wavelength range ofelectromagnetic radiation used for optical interrogation. In thenear-infrared wavelength range of the electromagnetic radiationspectrum, perfluorinated polymers such as fluorinated ethylene propyleneare preferable due to their low optical absorption, chemicalcompatibility, and classification as USP Class VI compliant materials.Alternatively, if polymers exhibiting substantial absorption in thewavelength range of interest are to be used, at least portions of thefirst 110 and second 120 surfaces in the optical sampling region 130 maybe manufactured to be sufficiently thin to provide sufficient opticalthroughput in the wavelength range of interest. For example,polycarbonate exhibits strong absorption features in the near-infraredwavelength range, however the transmission of polycarbonate isacceptable if the polycarbonate is sufficiently thin, and preferablyless than 0.25 mm thick. Due to the sterility requirements common inbioprocessing applications, polymers chosen for manufacturing of thebioprocessing vessel 100 and optical sampling region 130 will preferablybe amenable to sterilization by one or more techniques. Sterilization byirradiating with gamma or beta radiation is a common technique fordisposable polymer components in bioprocessing applications. Bothsterilant gas such as ethylene oxide or heat sterilization by autoclaveare also sterilization options, and embodiments will preferablywithstand sterilization by at least one sterilization technique andremain FDA and/or USP Class VI compliant after sterilization.

The sectional view in FIG. 2 shows the bioprocessing vessel of FIG. 1with an optical reader 150 configured to provide communication ofelectromagnetic radiation into and out of the fluid within the opticalsampling region 130. The optical reader 150 may comprise a housing 160and optical elements 170 such as waveguides or lenses to communicatelight to and from the optical sampling region 130. Electromagneticradiation from an optical instrument or light source and traveling inthe direction of arrowed line A may be communicated through one or moreoptical elements 170 in the optical reader 150, and through the firstsurface 110. A portion of the electromagnetic radiation havinginteracted with the fluid within the disposable bioprocessing vessel 100may be communicated through the second surface 120 and additionaloptical elements 170 within the optical reader 150. The optical reader150 may then communicate a portion of the resulting electromagneticradiation to the optical instrument or a separate sensor for sensing.

In an embodiment similar to the bioprocessing vessel 100 with featuresthat extend outward sideways from the primary volume of the vessel, anembodiment of a bioprocessing vessel 180 with features extending outwardthrough the bottom of the vessel are also provided. First 110 and second120 surfaces are provided and form an optical sampling region 130. Saidbioprocessing vessel 180 shown in FIG. 3 and FIG. 4 provides similarfunctionality to the bioprocessing vessel having features extendingoutward sideways 100. The positioning of the optical sampling region 130at the side or bottom of the bioprocessing vessel allows flexibility inthe configuration of the receiving and optical sampling components thatinterface with the vessel. The first 110 and second 120 surfaces neednot extend out from the primary volume of the disposable bioprocessingvessel in the shape depicted in FIG. 3 and FIG. 4, and indeed it may bepreferable to minimize the extent of the protrusion to ensure adequatemixing and fluid homogeneity. Baffles or other directional surfaces (notshown) may be provided to encourage fluid mixing within the samplingregion 130. In one embodiment, the first 110 and second 120 surfaces maycomprise step-variable features to provide more than one optical pathlength through the fluid. The ability to select from a plurality ofoptical path lengths is particularly advantageous in bioprocessingapplications where the turbidity of the fluid may change substantiallythroughout the process. For example, in a first part of a cell cultureor fermentation process where the turbidity is low, a longer opticalpath length may be chosen to increase the optical interaction lengthwith the fluid and substances contained therein. In a subsequent part ofthe process when the turbidity is high due to cell growth, a shorteroptical path length may be chosen to reduce the attenuation from thecells and thereby increase the optical signal.

Embodiments comprising disposable bioprocessing vessels with polymerregions for optical wavelength reference operations are provided by thepresent invention. Polymers for optical wavelength referencing may bethe same polymer as the primary polymer comprising the bioprocessingvessel, or may be a different polymer having more desirable propertiesfor wavelength reference operations. The disposable bioprocessing vessel180 embodiment shown in FIG. 4 comprises an additional polymer element185 configured for optical wavelength reference operations. Saidadditional polymer element 185 is configured such that an optical beamincident in the direction of arrowed line E may pass through said secondpolymer element 185 without traversing the fluid contained within thebioprocessing vessel 180. Such a configuration where an optical beamused for wavelength reference operations traverses only the referencepolymer and not any fluid within the bioprocessing vessel is preferableso that no optical signature from the fluid (which may change over time)is included in the wavelength reference operation.

Embodiments of the present invention comprising composite polymerlaminates are also provided. A disposable bioprocessing vessel orportion thereof may comprise a plurality of polymer layers adjacent toone another as shown in FIG. 5. Such a composite polymer 200 maycomprise for example a first 210, second 220, and third 230 polymerlayer. The composite polymer 200 may be formed by any number of methodsincluding layers that are co-extruded, fusibly bonded, adhesivelybonded, thermally bonded, ultrasonically bonded, or connected at seams.Such composite polymer laminates may find application where variousproperties are required of the bioprocessing vessel that cannot beeasily accomplished with a single polymer. For example, a first polymerbeing FDA or USP Class VI compliant may be used as a liner in contactwith the fluid contents of the vessel while a second polymer maycomprise an outer structural layer of the vessel, and need not be incompliance with FDA or USP guidelines as no surfaces of the secondpolymer are wetted. Use of a transition polymer between an inner and anouter polymer may be used to improve adhesion between the inner andouter polymers. Additional polymers beyond the inner and outer polymersmay also be used for example to affect oxygen permeability of thecomposite polymer structure.

The embodiment shown in FIG. 6 provides a transflection measurementconfiguration. Such a configuration enables collection ofelectromagnetic radiation having portions both having been reflectedfrom the fluid and materials contained therein as well as having beentransmitted through said fluid and materials contained therein. Theembodiment shown in FIG. 6 provides an extension of the disposablebioprocessing vessel 100 of FIG. 1 and FIG. 2 wherein the second surface120 further comprises a reflector 190. The reflector 190 may comprisefor example an optical element such as a mirror, a dielectric coating,or a metallic coating, and may or may not be attached to the secondsurface 120.

In another embodiment, features that extend inward into the disposablebioprocessing vessel are provided for optical interrogation of the fluidwithin the vessel. An embodiment shown in FIG. 7 provides a disposablebioprocessing vessel 240 comprising two optical access features 260extending inward into the interior of the vessel to form an opticalsampling region 250 having a fixed optical path length through thefluid. Optical elements such as mirrors or prisms mounted in a receiveror optical reader may be used to provide optical communication to thefluid within the bioprocessing vessel 240. In another embodiment shownin FIG. 8, a disposable bioprocessing vessel 270 comprising opticalaccess features 280 providing two or more optical path lengths isprovided. In this embodiment, the optical sampling region 290 comprisestwo sampling regions providing distinct optical path lengths, a firstoptical sampling region 300 and a second optical sampling region 310providing a longer optical path length than the first optical samplingregion 300. An optical beam traversing the first optical sampling region300 providing the shorter optical path length through the fluid isrepresented by dashed arrowed line C, and an optical beam traversing thesecond optical sampling region 310 providing the longer optical pathlength through the fluid within the bioprocessing vessel is representedby arrowed line D. As noted previously, provision of more than oneoptical path length provides flexibility in sampling for examplebioprocesses where cell growth occurs during a process and results inincreasing turbidity and hence increasing attenuation of optical beamstraversing the fluid.

Due to the fact that many polymers exhibit strong absorption features incertain wavelength ranges of the electromagnetic spectrum, it may beadvantageous to provide a second polymer serving as an optical window inthe optical sampling region of a disposable bioprocessing vessel. Forexample in the near-infrared wavelength range of the electromagneticspectrum (comprising wavenumbers between 3300 cm⁻¹ and 14,000 cm⁻¹),strong absorption features may arise from C—H, C—O, O—H, and N—Hchemical bonds. For this reason it may be preferable to use polymerslacking such chemical bonds in the optical sampling regions ofdisposable bioprocessing vessels designed for optical interrogation bysuch wavelengths. Perfluorinated polymers such as Teflon®polytetrafluoroethylene (PTFE), ethylene tetrafluoroethylene (ETFE),Teflon® fluorinated ethylene propylene (FEP), Teflon® amorphousfluoroplastics (AF), and Teflon® perfluoroalkoxy copolymer (PFA) lackthe listed chemical bonds and thus may be preferable for polymermaterials within the optical path. Alternatively, other polymermaterials may be used if sufficiently thin to provide adequate opticaltransmission in the desired wavelength range. An embodiment is providedthat comprises a second polymer being more optically transparent in thenear-infrared wavelength range than the primary polymer used in themanufacture of disposable bioprocessing vessels. Such an embodiment isshown in FIG. 9 and FIG. 10—a disposable bioprocessing vessel 320 havinga second polymer 330 in the optical sampling region 130 is provided forimproved optical transmission at near-infrared wavelengths of theelectromagnetic spectrum. A side view of such a vessel is shown in FIG.9 and a sectional view is shown in FIG. 10. The optical path lengthprovided in the optical sampling region 130 may be defined by the first110 and second 120 surfaces as with the previously described embodiment100, or the optical path length may be defined by the second polymer 330window region.

In yet another embodiment of the present invention, the optical pathlength may be formed by compression of the first 110 and/or secondsurfaces 120. The embodiment shown in FIG. 1 and FIG. 2 may provide suchan optical path length formed by compression. Compression of the first110 and/or second 120 surfaces may be provided by elements of theoptical reader 150 or by a receiver assembly. In this embodiment, atleast portions of the first 110 and second 120 surfaces are partiallycompressible such that they can be mechanically compressed to provide adefined optical path length. Compression of surfaces to provide a pathlength enables a highly stable and reproducible path length, which is ofconsequence for high-performance spectroscopic measurements.Establishing an optical path length by compression may also provide thebenefit of reducing the tolerances on manufacturing of the disposablebioprocessing vessels. The merits of establishing an optical path lengthby compression of polymer components have been established in U.S.patent application Ser. No. 14/631,914, the teachings of which areincorporated by this reference. If rigid surfaces in non-disposablecomponents of an optical reader or receiver are used to compresssurfaces on a disposable bioprocessing vessel to form a defined opticalpath length, tighter tolerances may be used in the manufacturing of thenon-disposable components as a higher manufacturing cost would beacceptable in non-disposable components whereas manufacturing costs aredesirably minimized in production of disposable components.

Embodiments of disposable bioprocessing vessels comprising integraloptical probes extending into the fluid within the bioprocessing vesselsare also provided by the present invention. Provision of optical probesenables alternative optical interfacing strategies to opticalinstrumentation and permits sampling the contents of bioprocessingvessels at locations more central to the vessel rather than at theperiphery. One embodiment of a disposable vessel 340 comprising anintegral optical probe 350 is shown in the isometric view in FIG. 11 andthe sectional view in FIG. 12. In this embodiment, optical waveguides360 such as optical fibers provide optical communication between opticalelements 370 situated within the bioprocessing vessel 340 and an opticalreader, sensor, or receiver for the bioprocessing vessel 340. An opticalsampling region is provided between the optical elements 370, and theoptical path length is determined by the distance between the opticalelements 370 within the fluid. Additional optical components (not shown)may be provided in the optical probe 350 to enhance optical throughputor otherwise improve performance or alter functionality. For example,lenses may be provided to image the optical beam through the opticalsampling region. It is preferable that all wetted surfaces to come incontact with the fluid in the bioprocessing vessel 340 be comprised ofpolymers or other suitable materials that are FDA and/or USP Class VIcompliant for bioprocessing applications. The integral optical probe 350may be located in alternative locations on the bioprocessing vessel 340such as through the top 380, bottom 390, or a sidewall 400.

The embodiment shown in FIG. 11 and FIG. 12 may be configured to providean optical transmission or transflection measurement. The distancethrough the fluid between the optical elements 370 forming the opticalpath length will preferably be between 0.5 mm and 2.0 mm (inclusively)for near-infrared spectroscopic measurements. This range of optical pathlengths is favorable when electromagnetic radiation having wavenumbersbetween 3300 cm-1 and 5600 cm-1 is employed in the optical measurementowing to sufficiently high optical interaction length with the fluid andsufficiently low water absorption.

Additional embodiments of disposable bioprocessing vessels havingintegral optical probes are shown in FIG. 13 and FIG. 14. Theseembodiments provide simple optical configurations to enable low-costmanufacturing and ease of alignment with supplemental opticalinstrumentation. In the embodiment shown in FIG. 13, a disposablebioprocessing vessel 410 is spanned by an optical probe 420 having anoptical sampling region 430 transverse to the axis of the probe 420. Afirst optical waveguide 440 within the optical probe 420 providesoptical communication between an optical instrument and the opticalsampling region 430 of the probe 420 where a defined optical path lengththrough the fluid is provided. Additional optical components may beprovided within the probe 420 to improve optical throughput or otherwisetailor the performance to an application. In this embodiment, a sensormay be placed directly below the disposable bioprocessing vessel 410within a receiver or the collected light having interacted with thefluid may be communicated to an optical instrument. In one preferredembodiment, a second optical waveguide 450 being larger in diameter thanthe first optical waveguide 440 is provided to collect light havinginteracted with the fluid in the optical sampling region 430. Saidsecond optical waveguide 450 may then communicate collected light to anoptical instrument or sensor. In this embodiment, if the optical pathlength remains sufficiently short (e.g. 2.0 mm or less) and the secondwaveguide 450 is sufficiently larger in diameter than the first opticalwaveguide 440, high optical throughput may be provided without theprovision of additional optical elements. This approach enablesreduction in the number of optical components required, thereby reducingmanufacturing costs and simplifying the optical geometry.

The embodiment shown in the sectional view in FIG. 14 provides adisposable bioprocessing vessel 460 with an optical sampling region 470formed between the end of the optical probe 490 and a wall of thebioprocessing vessel 460. In the embodiment shown in FIG. 14, theoptical sampling region 470 that forms the optical path length islocated between the end of the optical probe 490 and the bottom wall 480of the bioprocessing vessel 460. Similarly the optical probe 490 may beinstalled in different locations within the bioprocessing vessel 460 ifadvantageous for the application. In one embodiment, a single opticalwaveguide 500 within the optical probe 490 may be provided if theoptical path length provided by the optical sampling region 470 issufficiently short to maintain acceptable optical throughput. Opticaltransmission and transflection measurements may be provided by certainembodiments of the invention. For example, an optical transflectionmeasurement may be provided by including a reflective element on thebottom wall 480 opposite the optical probe 490 of the bioprocessingvessel 460.

Embodiments of receivers for disposable bioprocessing vessels are alsoprovided by the present invention. Receivers may accommodate a singlebioprocessing vessel, but are commonly configured to receive a pluralityof bioprocessing vessels to perform multiple bioprocessing experimentssimultaneously. Receivers may perform a plurality of functions such asmeasurement and/or control of temperature, agitation, aeration, pH,dissolved oxygen, cell density, cell viability, and chemical constituentconcentrations. One embodiment of a receiver is shown in the isometricview in FIG. 15. In this embodiment, the receiver 510 is configured toreceive a plurality of bioprocessing vessels 520. A base assembly 530 isprovided with a plurality of stations 540 configured for receivingbioprocessing vessels 520. Each station 540 within the receiver 510 isconfigured to receive a bioprocessing vessel 520, and may providecomponents for near-infrared optical interrogation of the contents ofthe bioprocessing vessel 520. Optical components such as optical fibers,lenses, mirrors, and sensors may be provided within the receiver baseassembly 530 in conjunction with each station 540 to provide opticalcommunication between the contents of the bioprocessing vessels 520 andone or more optical instruments. Optical communication betweenbioprocessing vessels 520 and optical instrumentation may also beprovided by an optical interface 560 configured with a mechanicaltranslator 570. In such an embodiment, one or more motors 580 may beused to translate the optical interface 560 on the mechanical translator570 to a desired bioprocessing vessel 520 where the optical interface560 performs optical interrogation on the fluid contents of thebioprocessing vessel 520. The optical interface 560 may comprise opticalelements such as fibers, lenses, and windows to provide opticalcommunication with bioprocessing vessels 520 and any associated featuresor integral optical probes. Similarly, the bioprocessing vessels 520 maybe mechanically translated and the optical interface 560 may remainstationary.

The view in FIG. 16 provides additional detail on an embodiment of thereceiver base assembly 530 and how interfacing with bioprocessingvessels 520 may be performed. With optical sampling, proper alignment ofthe bioprocessing vessels 520 within the receiver 510 is preferable toensure satisfactory alignment of optical components. Stations 540 withinthe receiver base assembly 530 may comprise specific alignment features550 such as a notch or groove to couple with a corresponding feature ona bioprocessing vessel 520 to ensure satisfactory alignment.Alternatively, features 590 that correspond to optical sampling featureson the bioprocessing vessel 520 may serve as the alignment means.

The sectional view in FIG. 17 offers detail on embodiments forinterfacing a receiver 510 with three embodiments of disposablebioprocessing vessels. From left to right in the figure, disposablebioprocessing vessels with: optical sampling features extending outwardsideways 100 (from FIG. 1 and FIG. 2); an integral optical probe forminga gap with the bottom wall of the vessel 460 (from FIG. 14); and anintegral optical probe having an optical sampling region transverse tothe axis of the probe 410 (from FIG. 13) are shown housed withinstations 540 in the receiver 510. Embodiments of the present inventionmay provide optical elements such as optical sensors 600 located withinthe base assembly 530 of the receiver 510. For example, the opticalwaveguide 500 provided in the optical probe 490 of the bioprocessingvessel 460 may provide optical communication of near-infraredelectromagnetic radiation from an optical source or instrument to theoptical sampling region 470 forming an optical path length in the fluid,and resultant electromagnetic radiation may be sensed by the sensor 600.

Methods are also provided by the present invention for determiningquantities of substances within fluids contained within disposablebioprocessing vessels. Near-infrared electromagnetic radiation may beused to optically interrogate fluids, and the changes sensed in thecollected near-infrared radiation after interaction with a fluid may beused to determine quantities of substances within fluids. Bioprocessingvessels located in a receiver assembly may first be selected for opticalinterrogation. Selection of a vessel may be performed for examplemechanically as by translating an optical interface located on amechanical translator, or optically as by activation of an opticalsensor or switch. Near-infrared electromagnetic radiation is thencommunicated to a disposable bioprocessing vessel. Communication ofnear-infrared radiation may be provided by optical waveguides such asoptical fibers, free-space optical elements, or a combination thereof.Optical communication elements may be provided in the receiver baseassembly, on an optical interface, or both. For example, near-infraredelectromagnetic radiation from an optical instrument may be communicatedto a bioprocessing vessel via optical waveguides, and radiation havinginteracted with the fluid within a bioprocessing vessel may be sensed byan adjacent optical sensor. Radiation having interacted with the fluidwithin a bioprocessing vessel may also be communicated to an opticalinstrument for analysis. Radiation resulting from optical transmissionor transflection measurements through the fluid in the bioprocessingvessel may be used by an optical instrument to determine one or morequantities of substances in a fluid.

Optical spectroscopy with near-infrared electromagnetic radiation offersa plurality of advantages for determining quantities of substances influids. Optical absorption features in the 3300 cm-1 to 14,000 cm-1wavenumber range are often present for substances having C—H, O—H, C—O,N—H, S—H, and P—H chemical bonds, offering the possibility to determinequantities of substances containing such chemical bonds usingnear-infrared spectroscopy. While water is sufficiently stronglyabsorbing in several wavelength ranges throughout the infraredelectromagnetic spectrum to limit the effectiveness of spectroscopictechniques to determine quantities of substances, the 3300 cm-1 to 5600cm-1 wavenumber range provides a water transmission window centered atapproximately 4600 cm-1. In this wavenumber range the water absorptionis sufficiently low to allow adequate optical throughput through fluidsamples with a sufficiently short optical path length to determinequantities of substances by spectroscopic techniques. In order toprovide sufficient optical throughput through a fluid and also provide asatisfactory optical path length for interaction of electromagneticradiation with the fluid, optical path lengths through fluids rangingfrom 0.5 mm to 2.0 mm are preferable for embodiments of the presentinvention. Measurements with near-infrared spectroscopic techniques maybe used to determine quantities of substances in fluids such asalcohols, sugars, lipids, organic acids, peptides, and steroidalmolecules as such substances often comprise optical absorption featuresat near-infrared wavelengths due to their chemical bonds. In addition tomeasurements of optical absorption by transmission or transflectionmeasurement approaches to determine quantities of substances by theirabsorption spectra, near-infrared spectroscopic techniques may be usedto determine parameters such as cell density, cell viability, orturbidity. Due to the reduction in optical scattering with increasingwavelength, optical path lengths between 0.5 mm and 2.0 mm may be usedeven when conducting high cell density bioprocesses such as Pichiapastoris fermentations. Use of wavenumbers higher than 5600 cm-1(shorter wavelength than 1.8 μm) often requires short path lengths oroperation with low cell density applications due to the increasedoptical scattering encountered and resulting optical attenuation.

Embodiments of the present invention including disposable bioprocessingvessels and receivers as well as associated methods provide for aplurality of bioprocessing applications such as a storage stage, agrowth stage, a product formation stage, a purification stage, and aproduct formulation stage. For example, a growth stage may include cellculture, fermentation, or other bioprocesses whereby cell growth and/orproduct formation is desired. Embodiments of the present invention maybe provided for processes such as batch processes as well as continuousprocesses such as perfusion processes. Downstream processes such asproduct purification may also utilize embodiments of the presentinvention for determination of constituents in fluids.

Embodiments of the present invention disposable bioprocessing vesselsmay also comprise polymer regions to provide an optical wavelengthreference. The merits of providing polymer materials for wavelengthreference operations have been described in U.S. patent application Ser.No. 14/631,917, the teachings of which are incorporated by thisreference. Absorption features of polymers may be used advantageously asoptical wavelength references, wherein said absorption features are usedto provide a comparison of a measured optical spectrum of the polymerwith a known optical spectrum of the polymer to determine the wavelengthaccuracy of an instrument. Such wavelength reference methods may provideenhanced stability of optical systems and measurements due to theestablishment of a calibrated wavelength axis of a measurement.Instrumental drift due to for example drift in performance of instrumentcomponents or in environmental conditions may cause undesirable changesto an optical system whereby the wavelength axis of the measurement maydeviate from an acceptable condition. Periodic verification andcorrection of the wavelength properties of an optical system bycomparison with a known standard material is desirable to mitigateagainst such undesirable changes and thereby improve the stability ofthe optical system and accuracy of measurements made with the opticalsystem. In embodiments of the present invention, a second beam ofnear-infrared electromagnetic radiation may be provided to opticallyinterrogate a polymer region on a disposable bioprocessing vesselcomprising a polymer suitable as an optical wavelength reference. Saidpolymer region will desirably provide no fluid sample within the secondoptical beam path such that the optical absorption experienced by thebeam is only that of the polymer wavelength reference material. Saidpolymer used as a wavelength reference material will desirably havemultiple optical absorption features within the wavelength range of theoptical measurement in order to provide multiple features with which tomake a comparison against a known optical spectrum of the polymer. Inthe near-infrared region of the electromagnetic spectrum, polymermaterials such as nylon, polycarbonate, Kapton®, polymethylpentene(TPX), and polyether ether ketone (PEEK) may be provided as wavelengthreference materials.

The present invention has been described with reference to the foregoingspecific implementations. These implementations are intended to beexemplary only, and not limiting to the full scope of the presentinvention. Many variations and modifications are possible in view of theabove teachings. The invention is limited only as set forth in theappended claims. All references cited herein are hereby incorporated byreference to the extent not inconsistent with the disclosure herein.Unless explicitly stated otherwise, flows depicted herein do not requirethe particular order shown, or sequential order, to achieve desirableresults. In addition, other steps may be provided, or steps may beeliminated, from the described flows, and other components may be addedto, or removed from, the described systems. Accordingly, otherimplementations are within the scope of the following claims. Anydisclosure of a range is intended to include a disclosure of all rangeswithin that range and all individual values within that range.

1. A disposable bioprocessing vessel for containing a fluid sample, thevessel comprising: at least one rigid wall; an optical sampling regionintegral to the rigid wall and comprising a first and second surface tocreate an optical path between the first and second surfaces within thebioprocessing vessel, said first and second surfaces comprise a polymerat least partially transparent to near-infrared electromagneticradiation and wherein the first and second surfaces are sufficientlythin to allow near-infrared electromagnetic radiation to passtherethrough, interact with the fluid sample, and be detected outside ofthe bioprocessing vessel to provide a transmission or transflectionmeasurement of the fluid sample.
 2. The disposable bioprocessing vesselof claim 1, wherein said polymer comprises at least one of polycarbonateor fluorinated ethylene propylene (FEP).
 3. The disposable bioprocessingvessel of claim 2, wherein at least portions of the first and secondsurfaces are less than 0.25 mm thick.
 4. The disposable bioprocessingvessel of claim 1, wherein said polymer comprises a composite polymerlaminate comprising a first layer and a second layer wherein the secondlayer comprises a different polymer material than the first layer. 5.The disposable bioprocessing vessel of claim 1, wherein a length of theoptical path defined by said first and second surfaces is between 0.5 mmand 2.0 mm inclusively.
 6. The disposable bioprocessing vessel of claim1, wherein said disposable bioprocessing vessel is suitable forsterilization by one or more of gamma irradiation, beta irradiation,ethylene oxide, or autoclave.
 7. The disposable bioprocessing vessel ofclaim 1, wherein said second surface further comprises a reflector,wherein said reflector reflects at least a portion of said near-infraredelectromagnetic radiation in the direction of the first surface where itmay be sensed by a sensor, thereby providing an optical transflectionmeasurement.
 8. The disposable bioprocessing vessel of claim 1, whereinsaid polymer is at least partially transparent to near-infraredelectromagnetic radiation transmitted through a fluid sample comprisingwavenumbers between 3300 cm⁻¹ and 5600 cm⁻¹.
 9. The disposablebioprocessing vessel of claim 1, wherein said disposable bioprocessingvessel has a working volume less than or equal to 0.25 liters.
 10. Thedisposable bioprocessing vessel of claim 1, wherein said first andsecond surfaces extend outward from a primary volume of saidbioprocessing vessel and define said optical path.
 11. The disposablebioprocessing vessel of claim 1, wherein said first and second surfacescomprise one or more features extending inward into a primary volume ofsaid bioprocessing vessel and defining said optical path therebetween.12. The disposable bioprocessing vessel of claim 1, wherein saiddisposable bioprocessing vessel is configured for a process selectedfrom the group consisting of a storage stage, a growth stage, a productformation stage, a purification stage, and a product formulation stage.13. The disposable bioprocessing vessel of claim 1, wherein said firstand second surfaces comprise regions with step-variable distancestherebetween, thereby providing a plurality of optical paths ofdifferent optical path lengths.
 14. The disposable bioprocessing vesselof claim 1, wherein said first and second surfaces comprise a secondpolymer, wherein the second polymer is more optically transparent thansaid polymer to near-infrared electromagnetic radiation, wherein atleast a portion of said second polymer is within said optical path. 15.The disposable bioprocessing vessel of claim 14, wherein said secondpolymer comprises fluorinated ethylene propylene (FEP).
 16. Thedisposable bioprocessing vessel of claim 1, wherein said vessel furthercomprises a region comprising a second polymer and at least a portion ofsaid region does not surround a fluid sample within the disposablebioprocessing vessel, wherein said second polymer provides opticalabsorption features to enable an optical wavelength reference.
 17. Thedisposable bioprocessing vessel of claim 1, wherein at least portions ofsaid first and second surfaces are at least partially compressible, andwherein a length of an optical path through the fluid sample is definedby compression of said first and second surfaces.
 18. A disposablebioprocessing vessel with features for optically sampling a fluid withinsaid bioprocessing vessel, said bioprocessing vessel comprising: a top,a bottom, and at least one rigid sidewall, wherein the top, bottom, andrigid sidewall define an interior; an optical probe integral with atleast one of the top, bottom, or rigid sidewall and protruding into theinterior of said disposable bioprocessing vessel, said integral opticalprobe comprising at least one optical waveguide; wherein a portion ofsaid integral optical probe is within the interior of said disposablebioprocessing vessel and provides optical communication between a fluidsample within said disposable bioprocessing vessel and an opticalinstrument whereby a near-infrared transmission or transflectionmeasurement is provided.
 19. The disposable bioprocessing vessel ofclaim 18, wherein the probe further comprises two optical elementspositioned to provide an optical path length therebetween and within theinterior of the bioprocessing vessel, whereby an optical transmission ortransflection measurement through the fluid sample contained within theinterior of said disposable bioprocessing vessel is provided.
 20. Thedisposable bioprocessing vessel of claim 18, wherein said integraloptical probe intersects two surfaces defining the interior of saiddisposable bioprocessing vessel, whereby near-infrared electromagneticradiation is communicated through one of said surfaces, interacts with afluid sample within said disposable bioprocessing vessel, and at least aportion of near-infrared electromagnetic radiation having interactedwith said fluid sample is communicated through the other of saidsurfaces thereby providing an optical transmission measurement.
 21. Thedisposable bioprocessing vessel of claim 18, wherein the length ofoptical path through said fluid is between 0.5 mm and 2.0 mminclusively.
 22. The disposable bioprocessing vessel of claim 18,wherein said disposable bioprocessing vessel has a working volume lessthan or equal to 0.25 liters.
 23. A receiver assembly for receiving oneor more disposable bioprocessing vessels and optically interrogatingsaid one or more disposable bioprocessing vessels with near-infraredelectromagnetic radiation, said receiver assembly comprising: a baseassembly comprising one or more stations each configured to receive adisposable bioprocessing vessel comprising components for near-infraredoptical interrogation, each of said one or more stations comprisingfeatures for alignment of said disposable bioprocessing vessels withinsaid stations such that said bioprocessing vessels may be installed inonly one orientation; one or more optical assemblies for opticalcommunication of near-infrared electromagnetic radiation between saiddisposable bioprocessing vessels positioned within said stations and anoptical instrument; wherein said optical assemblies for opticalcommunication comprise one or both of an optical interface notintegrated within said base assembly to provide optical communicationwith said disposable bioprocessing vessels or at least one opticalassembly integrated within said base assembly.
 24. The receiver assemblyof claim 23, further comprising a mechanical translator configured toprovide mechanical translation between said base assembly and saidoptical interface.
 25. The receiver assembly of claim 23, wherein saidreceiver assembly is configured to receive a plurality of disposablebioprocessing vessels.
 26. The receiver assembly of claim 23, whereinsaid stations are configured to receive disposable bioprocessingvessels, and wherein the disposable bioprocessing vessels compriseworking volumes less than or equal to 0.25 liters.
 27. The receiverassembly of claim 23, wherein said optical interface comprises one ormore optical waveguides to communicate near-infrared electromagneticradiation between an optical instrument and the disposable bioprocessingvessel.
 28. The receiver assembly of claim 23, wherein said receiverassembly further comprises one or more optical sensors.
 29. A method ofdetermining the quantities of one or more substances in a fluid samplecontained within a disposable bioprocessing vessel that is located in areceiver assembly, said method comprising the steps of: selecting adisposable bioprocessing vessel; communicating near-infraredelectromagnetic radiation from an optical instrument to said disposablebioprocessing vessel; collecting near-infrared electromagnetic radiationhaving interacted with a fluid content of said disposable bioprocessingvessel and communicating said electromagnetic radiation to said opticalinstrument for analysis; determining the one or more quantities ofsubstances in the fluid sample in said disposable bioprocessing vesselby utilizing the optical instrument to perform an optical transmissionor transflection measurement.
 30. The method of claim 29, wherein saidoptical instrument is configured to measure one or more of alcohols,sugars, lipids, organic acids, peptides, steroidal molecules, orproteins.
 31. The method of claim 29, wherein said optical instrument isconfigured to measure one or more of cell density, cell viability, orturbidity.
 32. The method of claim 29, wherein the step of determiningthe one or more quantities of substances is performed in a plurality ofbioprocessing vessels.
 33. The method of claim 29, wherein said methodfurther comprises the step of communicating a second beam ofnear-infrared electromagnetic radiation through a portion of saiddisposable bioprocessing vessel and not interacting with a fluid sample,wherein the optical absorption features of said polymer provide anoptical wavelength reference.